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Enzyme

Chymotrypsin

Chatchawin Petchlert, Ph.D.


Department of Biochemistry Faculty of Science, Burapha University

OUTLINE
xGENERAL PROPERTIES xCOFACTORS xCHEMISTRY OF CATALYST xENZYME KINETICS xENZYME INHIBITION xENZYME REGULATIONS

Binding of a substrate to an enzyme at the active site. The enzyme chymotrypsin, with bound substrate in red (PDB ID 7GCH). Some key active-site amino acid residues appear as a red splotch on the enzyme surface.

Enzyme

Ribozyme

Enzyme-substrate complex

Abzyme

History of Enzymology

Kuhne, 1878 Enzyme in yeast


bean

Buchner, 1897 Sumner, 1926 urease Jack Moore Stein, 1963 (amino
acid sequence) ribonuclease

Phillips, 1965 (three


dimensional structure) lysozyme
5

GENERAL PROPERTIES
t DEFINITION

Enzyme : a biological catalyst with * small amount * * G K * high efficiency and high specificity *
e q

Almost all enzymes are globular proteins.

10-5 M 10-4 10-3 M Turnover number (S) (P) 8

(Free energy change, G) (Equilibrium constant, Keq ) k1


constant S
k-1

k = rate

equilibrium

k1 = 10-3 min-1 , k-1 = 10-5 min-1 vf = [P] = k1 [S] 10-3 vr = k-1 [P] k
[S] k-1
1

Keq =

10-5

= 100

k 103 [P] 1 k1 k-1 106 [S] k-1 10

100

Keq =

(High specificity) 2 - (substrate specificity) - (reaction specificity)

10 - (chemical catalyst)

E (enzyme) + S (substrate)

Overall reaction
Binding

Step I :

ES (enzyme-substrate complex)

Step II : Transformation

E + P (product)

1. Absolute specificity

E S 2. Relative specificity E S Proteolytic enzyme t ENZYME NOMENCLATURE Trivial name refer reaction and/or substrate specificity Hexokinase, glucose phosphotransferase Systematic name 4 (E.C. number) 1.4.3.4 Monoamine : O2 oxidoreductase The 1st number : Class The 2nd figure : Subclass The 3rd figure : Sub-subclass The 4th figure : Serial number of the enzyme in the subsubclass

t SPECIFICITY OF ACTION

6 oxidoreductase dehydrogenase, transferase, hydrolase, lyase, isomerase ligase 3 (trivial name) (systematic name) 1 : 2 ase lactate pyruvate NAD+ 2 13

14

6 1 Oxidoreductase (oxidation-reduction) (redox reaction) Aox + Bred Ared + Box

2 Transferase 15

() 3 Hydrolase (hydrolysis) AB + H2O AOH + BH

4 Lyase 16 ABC AB + C

() 5 Isomerase ABC BAC

6 Ligase 2 ATP 17

COFACTORS
COFACTOR : Non-protein substances for optimum activity
with loosely bound or covalently bound

PROSTHETIC GROUP : cofactor when covalently bound,


become a permanent part of enzyme molecule

INORGANIC IONS : Zn2+, Mg2+, Mn2+, Fe2+ COENZYME : organic substance - water soluble vitamin

COFACTORS
(Protein~cofactor) (optimally active catalyst) dissociation is variable ; inactive or less active) ease of

HOLOENZYME

Protein (apoenzyme

Cofactor (inorganic ion or

organic substance ; inactive as a catalyst)

t ACTIVE SITE

Enzyme ACTIVE SITE Binding site Catalytic site Binding site : substrate ionic bond, H-bond, Van de Waals forces, hydrophobic interaction Catalytic site : binding site

ACTIVE SITE 1.

Active site

3. Substrate active site non covalent bonding active site substrate 4. active site substrate active site substrate

2. Active site 3

Stereo Specificity (Ogston, 1948)


Three point attachment 3 3

sp3

B
Enzyme surface

The tetrahedral structure of carbon orbital has rigid steric strain which makes the basic building unit of protein conformation

These two triangles are not identical


Juang RH (2004) BCbasics

t ENZYME SPECIFICITY

Model
enzyme substrate
1. Lock and Key Model - Fischer (1890) E-S 2. Induced Fit Model - Koshland (1958) E change 3. Strain or Transition State Stabilization Model - Haldane (1930), Pauling (1948) S change

Induced fit model

Enzymes in action 2: transition state stabilisation


Paulings Hypothesis I think that enzymes are molecules that are complementary in structure to the activated complexes of the reactions that they catalyse, that is, to the molecular configuration that is intermediate between the reacting substances and the products of reaction for these catalysed processes. The attraction of the enzyme molecule for the activated complex would thus lead to a decrease in its energy and hence to a decrease in the energy of activation and to an increase in the rate of reaction. Linus Pauling
Nature 161, 707 (1948)

(Binding energy) (strain) (Distortion) 27

The Nature of Enzyme Catalysis

Enzyme provides a catalytic surface This surface stabilizes transition state Transformed transition state to product
B A A B
Catalytic surface
Juang RH (2004) BCbasics

Enzyme Stabilizes Transition State


Energy change
Energy required (no catalysis)

ST
Energy decreases (under catalysis)

S ES

EST

EP
Reaction direction T = Transition state

Whats the difference?


Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.166

Active Site Is a Deep Buried Pocket


Why energy required to reach transition state is lower in the active site?
It is a magic pocket

+
CoE (1) (4) (3) (2)

(1) Stabilizes transition (2) Expels water (3) Reactive groups (4) Coenzyme helps
Juang RH (2004) BCbasics

Stickase
Substrate Transition state Product

If enzyme just binds substrate then there will be no further reaction

Enzyme not only recognizes substrate, but also induces the formation of transition state
Adapted from Nelson & Cox (2000) Lehninger Principles of Biochemistry (3e) p.252

Enzyme Active Site Is Deeper than Ab Binding

Instead, active site on enzyme also recognizes substrate, but g binding site on Ab binds to Ag actually complementally fits the mplementally, no further reaction transition state and stabilized it. curs.

Adapted from Nelson & Cox (2000) Lehninger Principles of Biochemistry (3e) p.252

Active Site Avoids the Influence of Water

+ -

ng the influence of water sustains the formation of stable ion


Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.115

Enzyme Kinetics

Enzyme Kinetics
Enzyme activity
Student A


Student C

Score

Student B

1 2 3 4 Exam Chapters

0 1 2 3 4 Substrate concentration

Increase the substrate concentration, observe the change of enzyme activity


Juang RH (2004) BCbasics

36

Invertase (IT)
Non-reducing sugar

Sucrose
HOCH2 O
1 2

IT

Reducing sugars

Reducing Power

Glucose + Fructose
HOCH2 6 O
4 5 1 2 OH

HOCH2 HOCH2 O HO H2O

HOCH2 HOCH2 6 1 O
5 2

OH

1 2 3 4 5 6

3 OH

CHO H-C-OH HO-C-H H-C-OH H-C-OH H2-C-OH

H2C-OH HO-C-H H-C-OH H-C-OH H2-C-OH


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HOCH2 O

HOCH2 HOCH2 O O

C=O

Increase Substrate Concentration

80 60

S + E P
(in a fixed period of ti

Product

40 20 0

Substrate ( mole)

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Essential of Enzyme Kinetics

Steady State Theory

E+

+P

In steady state, the production and consumption of the transition state proceed at the same rate. So the concentration of transition state keeps a constant.
Juang RH (2004) BCbasics

Constant ES Concentration at Steady State S


Concentration

ES
Reaction Time
Juang RH (2004) BCbasics

An Example for Enzyme Kinetics (Invertase)


1) Use predefined amount of Enzyme E 2) Add substrate in various concentrations S (x) 3) Measure Product in fixed Time (P/t) vo (y) 4) (x, y) plot get hyperbolic curve, estimate Vmx a 5) When y = 1/2 Vmx calculate x ([S]) Km a 1 vo
-1 Km 1 Vmx a

Double reciprocal 1/S

Km Direct plot S

Juang RH (2004) BCbasics

Vmx a vo 1/2

A Real Example for Enzyme Kinetics


Data
Substrate Product no Velocity Double reciprocal

1 2 3 4

[S] Absorbancev (mole/min) 0.25 0.21 0.42 0.50 0.36 0.72 1.0 0.40 0.80 2.0 0.46 0.92

1/S 4 2 1 0.5

1/v 2.08 1.56 1.35 1.16

(1) The product was measured by spectroscopy at 600 nm for 0.05 per mole (2) Reaction time was 10 min

Double reciprocal

Direct plot

1.0 v 0.5

2.0 1/v 1.0

1.0 -3.8
-2 0 2 4
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[S]

-4

1/[S]

Enzyme Kinetics
kcat / Km kcat
Turn over number

Vmx [S] vo= a Km + [S]

Direct plot

Significance

zero order 1st order E3 E2 E1

Observe vo change under various [S], resulted plots yield Vmax and Km

k3 [Et]
Activity Unit

Vmx a
Maximum velocity

&

Km
Competitive

Affinity with Double reciprocal substrate Inhibition

1 mole min
Specific Activity

Non-competitive

unit mg

Activity

Bi-substrate reaction also follows M-M equation, but one of the substrate should be saturated when estimate the other

Uncompetitive

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Significance of Enzyme Kinetics

Obtain

Vmx a

and

Km

v0 = Vmax K

= k3 [Et] K

Vmx [S] a vo = Km + [S]


Proportional to enzyme concentration

zero order

1st order [S] = Low High

E3 E2 E1

[S] = Fixed concentration


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Km: Affinity with Substrate

Vmx [S] a vo = Km + [S]


When using different substrate

Vmx a If vo = 2 Vmx Vmx [S] a a = 2 Km + [S]


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Vmx a S1
1/2

S2 S3

Km + [S] = 2 [S]
S1 S2 S3

Km

Affinity changes

Km = [S]

Km: Hexokinase Example

Glucose + ATP Glc-6-P + ADP


number CHO 1 H-C-OH 2 HO-C-H 3 H-C-OH 4 5 H-C-OH H2-C-OH 6

Glucose

Allose
CHO H-C-OH H-C-OH H-C-OH H-C-OH H2-C-OH

S Mannoseubstrate
CHO HO-C-H HO-C-H H-C-OH H-C-OH H2-C-OH

Km =

8,000

5 M
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Turn Over Number, kcat

E+S

k1 k2

ES (v ) E + P
o

k3

When substrate excess, k3 = kcat , turn over number (t.o. (IV)

When [substrate] is low

Second orde

k3 Vmx [S] k3 [E][S] a [E][S] vo = = = Km + [S] Km + [S] Km


Start from M-M eq.
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Omit the [substrate]Substrate specifici

Turn Over Numbers of Enzymes


Enzymes Catalase Carbonic anhydrase Acetylcholinesterase -Lactamase Fumarase Substrate
H2O2 HCO3Acetylcholine Benzylpenicillin Fumarate

kcat (s-1 ) 40,000,000 400,000 140,000 2,000 800 0.4

RecA protein (ATPase) ATP

The number of product transformed from substrate by one enzyme molecule in one second
Adapted from Nelson & Cox (2000) Lehninger Principles of Biochemistry (3e) p.263

Chymotrypsin Has Distinct kcat / Km to Different Substrates



O R O H3CCNCCOCH3

H H

R= Glycine Norvaline Norleucine H CH2CH2CH3 CH2CH2CH2CH3

kcat / Km
1.3 10-1 3.6 102 3.0 103 1.0 105 (M-1 s-1 )
Adapted from Mathews et al (2000) Biochemistry (3e) p.379

Phenylalanine CH2

Enzyme Activity Unit

t vo = [P] / min

Product [P]

S P mole

Slope tan 0 10 20 30 40
Juang RH (2004) BCbasics

Unit = mo /min
le Specific Activity Units Activity = Protein (mg)

Reaction time (min)

y x

y = tan x

Enzyme Inhibition (Mechanism)

I I U Competitive Ion-competitive ncompetitive N

Equation and Description Cartoon Guide

Substrate

E
S
I

X
I

Compete for Inhibitor active site

Different site

E + S ES E + P + I EI
[I] binds to free [E] only, and competes with [S]; increasing [S] overcomes Inhibition by [I].

E + S ES E + P + + I I EI + S EIS
[I] binds to free [E] or [ES] complex; Increasing [S] can not overcome [I] inhibition.

E + S ES E + P + I E IS
[I] binds to [ES] complex only, increasing [S] favors the inhibition by [I].
Juang RH (2004) BCbasics

Enzyme Inhibition (Plots)

I I U Competitive Ion-competitive ncompetitive N


vo
Vmx a

Direct Plots

vo

Vmx a

Vmx a

Vmx a

Vmx a

Km Km

[S], mM

Km = Km

[S], mM

Km Km

[S], mM

Double Reciprocal

Vmx unchanged a Km increased 1/vo


Intersect at Y axis

Vmx decreased a Km unchanged 1/vo

Both Vmx & Km decreased a 1/vo


Two parallel lines

1/ Vmx a 1/[S]

Intersect at X axis

1/ Vmx a 1/[S] 1/Km

1/ Vmx a 1/[S]

1/Km

1/Km

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Competitive Inhibition
Product Substrate Succinate C-OOC-H C-H C-OOC-OOH-C-H H-C-H C-OOCompetitive Inhibitor Glutarate C-OOH-C-H H-C-H H-C-H C-OOMalonate C-OOH-C-H C-OOOxalate C-OOC-OO-

Succinate Dehydrogenase
Adapted from Kleinsmith & Kish (1995) Principles of Cell and Molecular Biology (2e) p.49

Sulfa Drug Is Competitive Inhibitor


Domagk (1939)

Para-aminobenzoic acid (PABA)

H2NPrecursor

-COOH

Bacteria needs PABA for the biosynthesis of folic acid

Folic acid

Tetrahydrofolic acid

H2N-

-SONH2

Sulfa drugs has similar structure with PABA, and inhibit bacteria growth.

Sulfanilamide Sulfa drug (anti-inflammation)


Adapted from Bohinski (1987) Modern Concepts in Biochemistry (5e) p.197

Enzyme Inhibitors Are Extensively Used

Sulfa drug (anti-inflammation)


Pseudo substrate competitive inhibitor

Protease inhibitor

Alzheimer's disease

Plaques in brain contains protein inhibitor

HIV protease is critical to life cycle of HIV


HIV protease (homodimer): inhibitor is used to treat AIDS
subunit 1 subunit 2

Asp

Asp Symmetry

Human aspartyl protease:


(monodimer)

domain 1 domain 2Not

Asp

Asp

symmetry

Juang RH (2004) BCbasics

HIV protease vs Aspartyl protease


HIV Protease inhibitor is used in treating AIDS

HIV protease (homodimer) subunit 1


Asp Asp dimer

subunit 2 Symmetric

domain 1
Asp

domain 2
Asp

Asymmetric monomer

Aspartyl protease (monomer)


Juang RH (2004) BCbasics

Enzyme Catalysis

Chymotrypsin Catalytic Mechanism A1


Catalytic Triad
His57 Asp102 Ser195

H [HOOC] N C C

O C C O
C

N H

C N C

[NH2]

Check substrate specificity

Chymotrypsin Catalytic Mechanism A2


His57 Asp102

H
[HOOC] C N

H
N C H

Ser195

O
C C O

C N C

[NH2]

First Transition State

Chymotrypsin Catalytic Mechanism A3

H
[HOOC] C N

H
N C H

O
C O

C
C N

C C

[NH2 ]

Acyl-Enzyme Intermediate

Chymotrypsin Catalytic Mechanism D1

[HOOC]

C N

N-H C H
Acyl-Enzyme Water Intermediate

H O H

O
C O

C
C N

C C

[NH2 ]

Chymotrypsin Catalytic Mechanism D2

H H

Second Transition State

O
C O

C
C N

C C

[NH2 ]

Chymotrypsin Catalytic Mechanism D3

Deacylation

O
C C O C N C [NH2]

Chymotrypsin Is Activated by Proteolysis


Chymotrypsinogen (inactive) 245 Trypsin
Adapted from Campbell (1999) Biochemistry (3d) p.179

-Chymotrypsin (active) R15-I16

-Chymotrypsin S14-R15 L13 I16 Y146 T147-N148 A149 Disulfide bonds

-Chymotrypsin (active)

Asp 102

His 57

O COH

H N C NH

Active Ser
-

OCH2

Asp 102

C C H CH2 His 57

Ser 195

Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.158

Charge Relay in Active Site

H
-

CO

HN

HOCH2

C C H CH2

Ser 195

pH Influences Chymotrypsin Activity

Relative Activity 5 6 7 8 9 10 11

pH
Adapted from Dressler & Potter (1991) Discovering Enzymes, p.162

Buffer pH

Net Charge of a Protein

Juang RH (2004) BCbasics

pH Influences Net Charge of Protein

10 9 8 7
Isoelectric point, pI

6 5 4 3

Imidazole on Histidine Is Affected by pH


pH 6 HN C H C NH C H
O H HN C Inactive

pH 7 N C H

HN C

CO Asp 102

+ NH HOCH2

C C-H CH2 His 57

Ser 195

Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.158

Adapted from Dressler & Potter (2000) Discovering Enzymes, p.163

Chymotrypsin Produces New Ile16 N-Terminal


New NH2-terminus Relative activity

L13

I16

Y146

pH 5

9 10 11

NH2 Ile 16
Asp 194

CH2COO
+

pH 9

pKa

pH 10

NH3 Ile 16
Adapted from Dressler & Potter (1991) Discovering Enzymes, p.165

New Ile16 N-Terminal Stabilizes Asp194


Adapted from Dressler & Potter (1991) Discovering Enzymes, p.206

Catalytic Triad His 57 Asp 102 Ser 195 Asp 194 Gly 193

NH3 Ile 16

Nelson & Cox (2000) Lehninger Principles of Biochemistry (3e) p.112

Chymotrypsin Ser195 Inhibited by DIFP


Diisopropyl-fluorophosphate (DIFP) (CH3)2CHO P OCH(CH3)2 F

X
=
O CH2 O

(CH3)2CHO P OCH(CH3)2

O-H
CH2

Ser 195

Ser 195
Adapted from Dressler & Potter (1991) Discovering Enzymes, p.167

Addition of Substrate Blocks DIFP Inhibition


100 + DIFP Percent Inhibition of activity (%)

No substrate

X
50 + DIFP & substrate

Add substrat

Reaction time
Adapted from Dressler & Potter (1991) Discovering Enzymes, p.167

Chymotrypsin Also Catalyzes Acetate


Hartley & Kilby Nitrophenol acetate

O -C NH
Peptide bond

O CH3CO
+ H2O

NO2
Chymotrypsin

O -C OEster bond

Acetate

HO

NO2

O CH3COH

Nitrophenol

No acetate was detected at early stage


Adapted from Dressler & Potter (1991) Discovering Enzymes, p.168

Two-Stage Catalysis of Chymotrypsin


O CH3CO
Nitrophenol acetate

Acylation

NO2

OC

O CH3C

HO

NO2 Kinetics of reaction

O C
Deacylation (slow step)
Nitrophenol

CH3COOH

O-H C

+ H2O

Two-phase reaction

Time (sec)
Adapted from Dressler & Potter (1991) Discovering Enzymes, p.169

Extra Negative Charge Was Neutralized


-C-C-N-C-C-N-C-C-NH H O-C NHO H O-C NHO H

O -C NH

E+S

O -C-OH NH2Adapted from Dressler & Potter (1991) Discovering Enzymes, p.179

Active Site Stabilizes Transition State


Catalytic Triad
Ser 195 Asp 194 Met 192 His 57 Gly 193

Active Site
Asp 102 Cys 191 Thr 219 Cys 220

Ser 214 Trp 215

Specificity Site

Gly 216 Ser 217

Ser 218

Adapted from Dressler & Potter (1991) Discovering Enzymes, p.197

Acid-Base Catalysis
Adapted from Nelson & Cox (2000) Lehninger Principles of Biochemistry (3e) p.252

Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.167

Specific

Induced to transition state

Acid-base Catalysis O C H +

+
N H

Acid catalysis

+ O C H
O C H H C

N H

N H

C H

O H H

N H H

O C H
O C H H Base catalysis

N H H O

Both

N C H O H H H O C

O C H

Slow

Fast

Fast

Very Fast

Basic Mechanism of Catalysis


3 basic types 1) Bond Strain

Conformational chang 2) Acid-base transfer Chemical reaction 3) Orientation Space arrangement

Carboxypeptidase Anon-polar Metal protease Concert Carboxypeptidase B RK Exopeptidase non-specific Carboxypeptidase Y YFW RK GA

Chymotrypsin Sequential Trypsin Elastase

Ser-protease Endopeptidase

Juang RH (2004) BCbasics

Concerted Mechanism of Catalysis


Carboxypeptidase A Active site pocket (270) Glu 3 COO +

(248) Tyr O H
-

ACTIVE Site for SITE specificity

N
Substrate peptide chain

H O2

C
O-

C
COO -

R
+

5
Juang RH (2004) BCbasics

His (196)

Glu C-terminus (72) Check for His (69) C-terminal

Zn

Arg (145)

Chymotrypsin Has A Site for Specificity

O O NCCNCC NCCNCC R H R
OC Ser

Specificity Site Catalytic Site

Active Site
Juang RH (2004) BCbasics

Specificity of Ser-Protease Family


Trypsin
cut at Lys, Arg O O CNCCN C C C C NH3 + COOC Asp

Chymotrypsin
cut at Trp, Phe, Tyr O O CNCCN C

Elastase
cut at Ala, Gly O O CNCCN CH3
Shallow and non-polar pocket

Deep and negatively charged pocket

Non-polar pocket

Active Site

Juang RH (2004) BCbasics

Control Points of Gene Regulation


Transcription DNA
RNA Processing RNA Transport RNA Degradation

DNA
5 process mRNA 3 mature mRNA 3 tail proteins

ribosome mRNA

Translation
proteins

cap 5

Activity Proteolysis

Prokaryotics

Post-translational control

Eukaryotics
Juang RH (2004) BCbasics

Regulation of Enzyme Activity


Inhibitor Proteolysis
I
or
I

o
S

I
inhibitor

proteolysis
S

Feedback regulation
S

Phosophorylation

R regulator effector

P
S

o
(+) P

phosphorylation
A or
S
Juang RH (2004) BCbasics

Signal transduction

Regulatory subunit (-)

cAMP or calmodulin

Cascade Amplification of Signals

Cascade

nS

1 Enzyme

nP

Juang RH (2004) BCbasics

85

86

87

88

89

90

91

Two views of the regulatory enzyme aspartate transcarbamoylase (derived from PDB ID 2AT2). The regulatory clusters form the points of a triangle surrounding the catalytic subunits. Binding sites for allosteric modulators are on the regulatory subunits. Modulator binding produces large changes in enzyme conformation and activity.

92

Two views of the regulatory enzyme aspartate transcarbamoylase (derived from PDB ID 2AT2). This allosteric regulatory enzyme has two stacked catalytic clusters, each with three catalytic polypeptide chains (in shades of blue and purple), and three regulatory clusters, each with two regulatory polypeptide chains (in red and yellow). The regulatory clusters form the points of a triangle surrounding the catalytic subunits. Binding sites for allosteric modulators are on the regulatory subunits. Modulator binding produces large changes in enzyme conformation and activity.

93

Phosphorylation
Ser Thr Tyr (His)
Fischer, Kreb (1978)

OH

Kinase

phosphorylation
Phosphatase

Conformational Protein Change dephosphorylastion


Glycogen phosphorylase b Glycogen phosphorylase a

Inactive Active

Active Inactive
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Regulation of Blood Sugar


Cori & Cori (1947)

Signal Transduction

Tyrosine-kinase-linked receptor

Decrease

Insulin gh igh blood sugar Liver HormoneGlycogen Blood Pancreas

Glycogen synthase

Glucose

Glucagon ow blood sugar

Glycogen phosphorylase

GTP-protein-linked receptor

Increase
Juang RH (2004) BCbasics

Rapid Response to Blood Sugar Concentration


Glycogen phosphorylase Glycogen synthase

Enzyme activity

0 Very rapidly

4 6 8 Time (min) Not controlled in gene lev


Adapted from Stryer (1995) Biochemistry (4e) p.597

Glycogen Phosphorylase, GP
Glycogen
Glc-1-P Glc-6-P Glycolysis P P

Protein kinase A

GP kinase
ATP

Phosphatase

Glycogen phosphorylase a* Phosphorylase P

n-1
Glucose (-) Caffeine (-)

R T

6.2 cAMP

6.4
AMP (+) ATP (-) Glc-6-P (-)
Juang RH (2004) BCbasics

+
6.3

Glycogen phosphorylase b (inactive)

cAMP Controls Protein Kinase A Activity


Regulatory subunits A A A A cAMP A A A Catalytic subunits A Active kinase

Nucleus
P

CREB Activation

CREB DNA

Gene expression

Alberts et al (2002) Molecular Biology of the Cell (4e) p. 857, 858

cAMP Is the Second Messenger


Sutherland (1971)
Adenylate cyclase Receptor

Glucagon

Cyclase G protein
ATP cAMP A

G protein
Cyclic AMP (second messenger)

Protein Kinase A

Protein Kinase A
Cascade

GP Kinase

GP Kinase GP b

GP kinase

GP1 a
GP

Inactive Active

Glycogen

Glc-1-P

Juang RH (2004) BCbasics

Receptors on Cell Membrane


Gilman, Rodbell (1994) Adenylate cyclase

Glucagon

G-protein-linked Receptor
+ Signal

GTP

GTP

-GDP +GTP

GDP

GDP

G protein A
The third group: Ion-channel-linked Receptor

Glycogen breadkdown Insulin Enzyme-linked Receptor


+ Signal

Glycogen Synthase

ActivationP
P

kinase
P

active
Glycogen Synthase

Protein Phosphatase

Glycogen synthesis

SH2 domain

Juang RH (2004) BCbasics

GP kinase GP kinase P

Phosphatase

GP b

Glucagon

Glycogen synthase

GP a

Glycogen synthase

Glycogen

PKA

Protein phosphatase-1

Protein phosphatase-1 P Protein phosphatase inhibitor-1

active inactive

Protein phosphatase inhibitor-1 P


Adapted from Kleinsmith & Kish (1995) Principles of Cell and Molecular Biology (2e) p.217

Signal Transduction
Hormone Signal Receptor

G-protein

G
Cyclase

Transducer Effector Enzyme Effector Effect


Juang RH (2004) BCbasics

Allosteric Enzyme ATCase


Active relaxed form Carbamoyl phosphate Aspartate Carbamoyl aspartate

COOCOOCH2 O CH2 O H2N-CH2N-C-O-PO32-+HN-C-COON-C-COOATCase H H H H

- - -

Quaternary structure

ATP Feedback inhibition

CCC
R R R R R R

Catalytic subunits Regulatory subunits Catalytic subunits

CTP CTP

CTP CTP

CTP CTP

CCC

Inactive tense form


Juang RH (2004) BCbasics

- - -

CTP

Nucleic acid metabolism

vo
Sigmoidal curve

Sigmoidal Curve Effect

Noncooperative (Hyperbolic) ATP

CTP

Cooperative (Sigmoidal)

Positive effector (ATP) brings sigmoidal curve back to hyperbolic Negative effector (CTP) keeps

vo
Exaggeration of sigmoidal curve yields a drastic zigzag line that shows the On/Off point clearly

Off On [Substrate]

Consequently, Allosteric enzyme can sense the concentration of the environment and adjust its activity
Juang RH (2004) BCbasics

Mechanism and Example of Allosteric Effect


Kinetics
R = Relax (active)
S

Models
Allosteric site (+)

Cooperation
R
S

vo

R R
vo (+)
S

[S]

Allosteric site

Homotropic (+) Concerted Heterotropic (+) Sequential Heterotropic (-) Concerted


Juang RH (2004) BCbasics

A
(+)

T
S

T
vo

[S]

T = Tense (inactive)

I
(-)
[S]

(-)

Activity Regulation of Glycogen Phosphorylase


Covalent modification Covalent modification

AMP

ATP Glc-6-P Glucose Caffeine

Glucose Caffeine

A P

A P

A A
P

A P

Garrett & Grisham (1999) Biochemistry (2e) p.679

A P

A P

A P

GP kinase GP phosphatase 1

P P

A P

spontaneously

Non-covalent Non-covalent

Major Metabolic Pathway


STAGE 1 Glycolysis Macromolecule Unit molecule

Glc-6-P
P

Glc-1-P

Glycogen phosphorylase

Glycogen

Glucose

Kinase

digestion

Starch H O 4 H3-C-H 0 3 H3-C-OH 1 2 H2-C=O 1 1 H-COOH 2 0 O=C=O 2

Oxidation of Carbon

6 3
Change of carbon number

Mitochondria Oxidative phosphorylation


P
P

STAGE 3 Energy production STAGE 2 Unit molecule Key small molecule

CO2

Pyruvate

Pyruvate

Juang RH (2004) BCbasics

Acetyl CoA A

$ $ $
Citric acid cycle

ATP

NADH

H 2O

Operon Expression Regulated by Its Metabolites


S S
R

Upstream metabolite (S) inactivates repressor, and induces the expression Gene mRNA

Operator

RNA Polymerase

ON

P
R
Operator

P
R

Downstream metabolite (P) might bind and activate repressor, Then turns off the gene expression Gene

RNA Polymerase

OFF
Juang RH (2004) BCbasics

Cross Talk between Cells


Direct contact
Direct contact

Diffusion
Local paracrine
(R)
Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.833

Short ranged Long ranged

(S)

Signaling cell (S)

Receptor cell (R)

Synaptic

Endocrine
(S)

(R)

Blood

Neuron

impulse

(S)

(R)

How to Separate These Objects

Shape Size wood stone Density

2 3 4 5 6

7 8 9

10 11

12

cotton wood wood cotton stone wood stone cotton stone co

Size

2 3
6
7 8
Density

Shape
cotton

4
8

4 5 6
9
10 11

wood

7
12

stone

Sieving different sizes

Different sedimentation

Different rolling speed


Juang RH (2004) BCbasics

Basic Principles of Protein Purification

Cell
Small molecule

Homogenization

Organelle Cell Debris

Macromolecule
(Lipid)

Amino acid, Nucleic acid ProteinCarbohydrate Sugar, Nucleotides, etc Ammonium sulfate fractionation

Size

Charge

Polarity

Affinity

Ion exchange, Reverse phase Gel filtration, chromatography, Affinity Chromatofocusing, chromatography, SDS-PAGE, HIC, Disc-PAGE, Hydroxyapatite Ultrafiltration Salting-out Isoelectric focusing
Juang RH (2004) BCbasics